Method of preparing an extract potent in anti-inflammation and anti-platelet aggregation from Zingiber officinale and pharmaceutical compositions containing said extract

ABSTRACT

A method of preparing an extract from  Zingiber officinale , which is potent in anti-inflammation and anti-platelet aggregation, includes the following steps: a) preparing a crude liquid from rhizomes of ginger by extraction with an organic solvent or by distillation with steam; b) introducing the crude liquid to a reverse phase chromatography column, and eluting the column with water, a first eluent and a second eluent having a polarity weaker than that of the first eluent but stronger than that of chloroform, so that a first eluate resulting from elution of the first eluent and a second eluate resulting from elution of the second eluent are obtained; c) removing the first eluent from the first eluate by evaporation, so that a first concentrated eluate is obtained and is able to used as the potent extract; and d) removing the second eluent from the second eluate by evaporation, so that a second concentrated eluate is obtained and is able to used as the potent extract.

FIELD OF THE INVENTION

The present invention is related to a method of preparing an extractpotent in anti-inflammation and anti-platelet aggregation from Zingiberofficinale.

BACKGROUND OF THE INVENTION

Chinese crude drugs or spices eg. Zingiber officinale, Eugeniacaryophyllata, Allium sativum, have been used in medicine and inflavoring foods. Crude ginger is used as an anti-emetic and expectorant,an anti-tussive and accelerator of the digestive organs. Semi-dried oldcrude ginger is also used for stomachache, chest pain, low back pain,cough, common cold and as a cure for a form of edema being called“stagnate of water”. Zingerone is the major component which accounts forthe spicy character of ginger; gingerol and shogaol are other pungentcomponents in ginger. Gingerol has cardio-tonic action, suppresses thecontraction of isolated portal veins in mice, and modulates theeicosanoid-induced contraction of mouse and rat blood vessels. Shogaolexhibits pressor response. Both gingerol and shogaol are mutagenic,whereas zinger and zingerone have been found to exhibit antimutagenicactivity. Shogaol has inhibitory activity on the carrageenin-induced pawedema and platelet aggregation [U.S. Pat. No. 5,804,603, Background ofthe Invention].

Heretofore, many reports have shown that Zingiber officinale exhibitsvarious physiological activities. Typical examples include a cancermetastasis suppressing agent disclosed in Japan patent publication No.7-258104; a synthesis promoter for neurotropic factor, which iseffective for nerve deteriorative diseases such as Alzheimer's dementiaor Parkinson's disease, disclosed in Japan patent publication No.7-25777; an anti-rheumatic agent disclosed in Japan patent publicationNo. 6-293653, U.S. Pat. Nos. 5,494,668 and 5,683,698; an antimicrobialcomposition disclosed in Japan patent publication No. 6-227931; and ananalgesic composition disclosed in Japan patent publication No.6-107556. Ginger contains 1-4% essential oil (oleoresin). During thelast 45 years many chemical investigations have been carried out on theconstituents of the essential oil. Altogether more than 200 differentvolatiles have been identified in essential oil wherein thepharmacological activity is confined. The essential oil contains amixture of various terpenes as well as some other non-terpenoidcompounds. Although this is mostly speculative, the experimental dataand observations suggest that ginger inhibits both the cyclooxygenaseand lypoxygenase products, i.e. it can be a dual inhibitor of eicosanoidsynthesis. In all 56 patients (28 with rheumatoid arthritis, 18 withosteoarthritis and 10 with muscular discomfort) used powdered gingeragainst their afflictions. Amongst the arthritis patients more thanthree-quarters experienced, to varying degrees, relief in pain andswelling. All the patients with muscular discomfort experienced reliefin pain. None of the patients reported adverse effects during the periodof ginger consumption which ranged from 3 months to 2.5 years.(Srivastava and Mustafa; Medical Hypotheses; 1992; 39 342-348).

Non-steroidal anti-inflammatory drugs have three major actions, all ofwhich are related to inhibition of cyclo-oxygenase resulting indecreased formation of prostanoids. Firstly, an anti-inflammatory actionachieved by reduced production of vasodilator prostaglandins (PGE2,PGI2) which means less vasodilation and, indirectly less edema.Secondly, an analgesic effect achieved by reduced prostaglandinproduction (less sensitization of nociceptic nerve endings to theinflammatory mediators bradykinin and 5-hydroxytryptamine). Thirdly, anantipyretic effect which is probably due to a decrease in the mediatorPGE2 generated in response to inflammatory pyrogens, much asinterleukin-1. Since ginger inhibits prostanoid synthesis and alsoproducts of 5-lipoxygenase, its ameliorative effects in arthritis andmuscular discomforts could be related to reduced formation ofprostanoids and leukotrienes. Because of such a possibility a decreasein the carageenan-induced edema formation in the rat's paw after 3 g ofginger extract administration has been demonstrated and the potency ofthe extract in the acute inflammation test appears to be comparable tothat exhibited by acetyl salicylic acid reported in the same study.(Mascolo N. et al Journal of Ethnopharmocology 1989, 27, 129-140).

SUMMARY OF THE INVENTION

The present invention provides extracts from rhizomes of ginger whichshow an activity in an in vitro anti-platelet aggregation test and aninhibitory activity on the carrageenin-induced paw edema. The extractsare prepared by extracting rhizomes of ginger with an organic solvent(such as ethyl ether, acetone, methanol and ethanol) or supercriticalCO₂, or by steam distilling rhizomes of ginger to obtain a crude liquid,and subjecting said crude liquid to a reverse phase chromatography toobtain the extracts containing shogaols, gingerols and/ordehydrogingerdione.

DETAILED DESCRIPTION OF THE INVENTION

As introduced in the Background of the Invention, ginger has been usedfor anti-inflammation and pain relief.

The present invention is to provide an effective method of preparing aproduct potent in anti-inflammation and in anti-platelet aggregationfrom rhizomes of ginger. The potent product prepared in accordance withthe method of the present invention has a substantially constantcomposition, so that the pharmaceutical effects thereof are definite.

The effective method of preparing product potent in anti-inflammationand in anti-platelet aggregation from rhizomes of ginger according tothe present invention comprises the following steps:

a) preparing a crude liquid from rhizomes of ginger;

b) introducing the crude liquid to a reverse phase chromatographycolumn, and eluting the column with water, a first eluent and a secondeluent in sequence, said second eluent having a polarity weaker thanthat of the first eluent but stronger than that of chloroform, so that afirst eluate resulting from elution of the first eluent and a secondeluate resulting from elution of the second eluent are obtained;

c) removing the first eluent from the first eluate by evaporation, sothat a first concentrated eluate is obtained and is able to be used asthe potent product; and

d) removing the second eluent from the second eluate by evaporation, sothat a second concentrated eluate is obtained and is able to be used asthe potent product;

wherein step a) comprises steps i) to iv), or comprises step I), stepI′), or step I″), wherein said steps i) to iv) are:

i) shedding fresh rhizomes of ginger and filtering the resulting mixtureto obtain a filtrate and a residue;

ii) extracting the filtrate with a first organic solvent, recovering theresulting extraction solution of the first organic solvent, andevaporating the first organic solvent from the extraction solution toobtain a first concentrated extraction solution;

iii) extracting the residue with a second organic solvent, recoveringthe resulting extraction solution of the second organic solvent, andevaporating the second organic solvent from the extraction solution toobtain a second concentrated extraction solution; and

iv) combining the first concentrated extraction solution and the secondconcentrated extraction solution to obtain the crude liquid;

said step I) is:

I) extracting powder of dried rhizomes of ginger with the second organicsolvent, recovering the resulting extraction solution of the secondorganic solvent, and evaporating the second organic solvent from theextraction solution to obtain the crude liquid;

said step I′) is:

I′) steam distilling powder of dried rhizomes of ginger, andconcentrating the resulting distillate by evaporation to obtain thecrude liquid; and

said step I″) is:

I″) extracting powder of dried rhizomes of ginger with supercriticalCO₂, recovering the resulting extraction solution of the supercriticalCO₂, and evaporating CO₂ from the extraction solution to obtain thecrude liquid.

The product potent in anti-inflammation and in anti-platelet aggregationprepared according to the method of the present invention preferablycomprises 0-10 mg 6-shogaol per gram of the product, 1-150 mg 6-gingerolper gram of the product, and 0-40 mg 6-dehydrogingerdione per gram ofthe product.

The present invention also provides an anti-inflammation pharmaceuticalcomposition comprising a therapeutically effective amount of said crudeliquid prepared in step a) of the method of the present invention, as anactive ingredient, in admixture with a pharmaceutically acceptablecarrier or diluent for the active ingredient.

The present invention also provides a pharmaceutical composition for theinhibition of aggregation of platelets, which comprises atherapeutically effective amount of said crude liquid prepared in stepa) of the method of the present invention, as an active ingredient, inadmixture with a pharmaceutically acceptable carrier or diluent for theactive ingredient.

The present invention also provides an anti-inflammation pharmaceuticalcomposition comprising a therapeutically effective amount of saidproduct prepared according to the method of the present invention, as anactive ingredient, in admixture with a pharmaceutically acceptablecarrier or diluent for the active ingredient. Preferably, said productprepared according to the method of the present invention is the firstconcentrated eluate prepared in step c). Alternatively, said productprepared according to the method of the present invention is the secondconcentrated eluate prepared in step d).

The present invention also provides a pharmaceutical composition for theinhibition of aggregation of platelets, which comprises atherapeutically effective amount of said product prepared according tothe method of the present invention, as an active ingredient, inadmixture with a pharmaceutically acceptable carrier or diluent for theactive ingredient. Preferably, said product prepared according to themethod of the present invention is the first concentrated eluateprepared in step c). Alternatively, said product prepared according tothe method of the present invention is the second concentrated eluateprepared in step d).

Preferably, said first eluent is methanol, and said second eluent isacetone.

Preferably, step a) of the method of the present invention comprisessteps i) to iv).

Preferably, said first organic solvent is ethyl ether.

Preferably, said second organic solvent is acetone, methanol, ethanol ora combination thereof. More preferably, said second organic solvent isacetone.

Preferably, step a) of the method of the present invention comprisesstep I).

Preferably, step a) of the method of the present invention comprisesstep I′).

Preferably, step a) of the method of the present invention comprisesstep I″).

A suitable reverse phase chromatography column for use in the method ofthe present invention includes (but is not limited thereto) a reversephase chromatography column packed with a porous resin, for examplesDiaion HP-20 (Mitsubishi Co.), Sephadex LH-20 (Pharmicia Co.) and RP-18(Nacalai tesque Co.).

Without further elaboration, it is believed that the above descriptionhas adequately enabled the present invention. The following specificexamples are, therefore, to be construed as merely illustrative, and notlimitations on the remainder of the disclosure in any way whatsoever.

DETERMINATION OF ACTIVE INGREDIENTS

In the following examples, high performance liquid chromatography(abbreviated as HPLC) was used to determine the active ingredients ofthe products prepared therein. HPLC spectra were recorded on a HPLCinstrument (HPLC Shimadzu LC-10AT, Japan) using a Cosmosil 5C-18 column(250 mm×4.6 mm, packed with particles having 5 μm diameter) by anelution method. An HPLC sample was prepared by diluting an appropriateamount of a product with a mobile phase solution (hydrogencyanide:water=65:35, V/V) to 25 ml, and filtered with a 0.25 μmmembrane. The filtrate was introduced into the HPLC column, and elutedwith the mobile phase solution. An UV detector (Shimadzu SPD-6AV, Japan)was used to detect the absorption of the eluate at 230 nm.

EXAMPLE 1

2100 g of fresh rhizomes of ginger were shredded and filtered to obtaina filtrate and a residue. 500 ml of the filtrate was extracted with 500ml ethyl ether three times, the organic phase layers were separated fromthe aqueous phase layers, and combined. Ethyl ether was evaporated fromthe combined extraction solution in vacuo to obtain a concentrated ethylether extraction product (I-OE). The ginger residue was extract with3000 ml acetone three times, the extraction solutions were recovered byfiltration, and combined. Acetone was evaporated from the combinedextraction solution in vacuo to obtain a concentrated acetone extractionproduct (I-O) (14.5 g). To a reverse phase chromatography column 300mm×30 mm packed with 180 g Diaion HP-20 resin having a diameter of 500μ-800 μ 7 g of a mixture of the concentrate ethyl ether extractionproduct (I-OE) and the concentrated acetone extraction product (I-O) wasinjected. 1500 ml water, 2500 ml methanol, 2000 ml acetone and 2000 mlchloroform were used to carry out elution. The water eluate, methanoleluate, acetone eluate and chloroform eluate were collected separately,and concentrated in vacuo to obtain 0.27 g concentrated water eluate(I-OW), 1.45 g concentrated methanol eluate (I-OM), 2.68 g concentratedacetone eluate (I-OA), and 0.83 g concentrated chloroform eluate (I-OC).The amounts (mg) of 6-shogaol, 6-gingerol and 6-dehydrogingerdione pergram of the I-O, I-OM and I-OA determined by HPLC are listed in Table 1.

TABLE 1 Content (mg/g) I-O I-OM I-OA 6-shogaol 1.10 ± 0.14 1.15 ± 0.0  —6-gingerol 59.98 ± 0.99  103.37 ± 8.57  2.51 ± 0.89 6-dehydrogingerdione7.68 ± 0.42 8.94 ± 0.41 —

EXAMPLE 2

500 g of shade dried rhizomes of ginger were pulverized and theresulting powder was extracted with 30 L acetone trice (each time with10 L). The three extraction solutions were combined together afterfiltration, and then concentrated in vacuo to obtain 24 g ofconcentrated acetone extraction product (II-O). To a reverse phasechromatography column packed with 600 g Diaion HP-20 resin 20 g of theconcentrated acetone extraction product (II-O) was injected, which wasthen eluted with 4 L water, 6.5 L methanol, 15 L acetone and 5 Lchloroform in sequence. The water eluate, methanol eluate, acetoneeluate and chloroform eluate were collected separately, and concentratedin vacuo to obtain 2.5 g concentrated water eluate (II-OW), 7.1 gconcentrated methanol eluate (II-OM), 6.9 g concentrated acetone eluate(II-OA), and 3.5 g concentrated chloroform eluate (II-OC). The amounts(mg) of 6-shogaol, 6-gingerol and 6-dehydrogingerdione per gram of theII-O, II-OM and II-OA determined by HPLC are listed in Table 2.

TABLE 2 Content (mg/g) II-O II-OM II-OA 6-shogaol 1.98 ± 0.00 4.96 ±0.00 — 6-gingerol 43.06 ± 0.84  70.87 ± 1.85  2.54 ± 0.006-dehydrogingerdione 9.33 ± 0.85 19.15 ± 4.57  2.35 ± 0.28

EXAMPLE 3

10 Kg of shade dried rhizomes of ginger were pulverized and theresulting powder was steam distilled for five hours. The distillate wasconcentrated in vacuo to obtain 410 g of concentrated distillate(III-O). To a reverse phase chromatography column packed with 600 gDiaion HP-20 resin 20 g of the concentrated distillate (III-O) wasinjected, which was then eluted with 4.5 L water, 4.5 L methanol, 3 Lacetone and 5 L chloroform in sequence. The water eluate, methanoleluate, acetone eluate and chloroform eluate were collected separately,and concentrated in vacuo to obtain 0.03 g concentrated water eluate(III-OW), 14.5 g concentrated methanol eluate (III-OM), 0.85 gconcentrated acetone eluate (III-OA), and 0.2 g concentrated chloroformeluate (III-OC). The concentrated distillate (III-O) contains no6-shogaol, 6-gingerol and 6-dehydrogingerdione determined by HPLC.

EXAMPLE 4

10 g of powder of shade dried rhizomes of ginger was extracted with 1000ml acetone at 50° C. for two hours. The extraction solution wasseparated and concentrated in vacuo (40° C., 75 mmHg) to obtain aconcentrated acetone extraction product (IV-O). The color and viscosityof the product (IV-O) together with its yield are listed in Table 3.

EXAMPLE 5

10 g of powder of shade dried rhizomes of ginger was steam distilled,and the oily distillate after being separated from the aqueousdistillate was freeze dried to obtain an oily extract (V-O). The colorand viscosity of the oily extract (V-O) together with its yield arelisted in Table 3.

EXAMPLE 6

To 10 g of powder of shade dried rhizomes of ginger in a 250 mlextraction chamber CO₂ was introduced at a flow rate of 45 L/min,wherein the chamber pressure was controlled at 2500 to 4000 psia with ahigh pressure pump (Model No. EK-1, LEWA Co., US) and the chambertemperature was maintained at 35-60° C. with a heat exchanger (Model No.H-2410, HOTEC Co., US) and an exterior circulation system. Theextraction was stopped when the volume of CO₂ introduced reached 300 L,and a supercritical CO₂ extraction product (VI-O) was obtained afterevaporation of CO₂. The color and viscosity of the product (VI-O)together with its yield are listed in Table 3. The contents of pungentcomponents determined by HPLC are listed in Table 4.

TABLE 3 IV-O V-O VI-O L* 87.6 80.4 96.3 A* −9.1 −0.1 −9.6 B* 31.1 9.622.0 Viscosity (cPs) 15.6 11.8 12.1 Yield (%) 3.8 2.2 3.9 *the values ofL, A, and B were determined by using a Σ90 color measuring system,(Nippon Denshoku Inc, Co., Ltd., Japan), wherein L represents lightness,A is the red/green difference and B is the yellow/blue difference.

TABLE 4 Content (mg/g) VI-O 6-shogaol 17.30 ± 0.00 6-gingerol 26.29 ±0.00 6-dehydrogingerdione 19.20 ± 1.19

EXAMPLE 7

Antiplatelet Assay

Blood, collected from the marginal ear vein of rabbits was mixed withEDTA (100 mM) in a volume ratio of 14:1 and centrifuged at 90 g for 10min at room temperature to obtain platelet-rich plasma. The latter wasfurther centrifuged at 500 g for 10 min, the upper plasma-rich layer wasremoved therefrom, and the remaining bottom layer was suspended withTyrode's solution containing 2 mM EDTA but no calcium. The suspensionwas further centrifuged at 500 g for 10 min and the platelets weresuspended with Tyrode's solution without EDTA. After centrifugation atthe same conditions, the platelets were suspended with Tyrode's solutionhaving the following compositions (mM): NaCl (136.8), KCI (2.8), NaNCO₃(11.9), MgCl₂ (1.1), NaH₂PO₄ (0.33), CaCl₂ (1.0), glucose (1 1.2) andborine serum albumin (0.35%). Platelet numbers were determined with aCoulter Counter (Model ZM) and adjusted to 4.5×10⁸ platelets/ml.

TABLE 5 The inhibitory effects of ginger extracts on plateletaggregation induced by arachidonic acid and collagen^(a)) GingerConcentration for 50% inhibitory effect (μg/ml) extracts Arachidonicacid Collagen I-O 3.8 ± 0.8 5.5 ± 0.4 I-OM 1.7 ± 0.3 2.7 ± 0.4 II-O 3.1± 0.5 6.5 ± 1.2 II-OA 10.9 ± 3.2  21.8 ± 2.2  II-OC 6.9 ± 0.7 16.6 ±4.3  II-OM 2.0 ± 0.2 6.9 ± 2.4 ^(a))Platelets were incubated with gingerextracts or 0.5% DMSO (Control) at 37° C. for 3 min, then arachidonicacid (100 μM) or collagen (10 μg/ml) was added to trigger aggregation.Aspirin and Indomethacin are positive controls. The percentage ofinhibitory effect is calculated as follows: {[(degree of inhibition ofControl) - (degree of inhibition of ginger extract)]/(degree ofinhibition of Control)} × 100% Values are presented as mean ± S.E., n =3-6.

EXAMPLE 8

Evaluation of Inhibitory Activity on the Carrageenin-induced Paw Edema

Inhibitory activity on the carrageenin-induced paw edema was conductedaccording to the method reported by Winter, C. A. et al. (Winter C. A.et al., Proc. Soc. Exper. Biol. Med. 111: 544-547, 1962.). Male Wistarmice weighing 150±20 g without feeding for one night were injected atleft rear paws thereof with 0.1 ml of 1% carrageenin suspension followedby rubbing test samples or vehicle as control on the left rear pawsevenly (10 mg/paw). Three hours later, the volumes of the rear paws weredetermined by using a volume scanner (Cat. #7150, UGO Basil, Italy), andthe difference between the left rear paw and the right rear paw was usedan index of the carrageenin-induced paw edema.

TABLE 6 Inhibitory activity on the carrageenin-induced paw edema ofginger extracts Inhibitory activity on the Dosage carrageenin-inducedpaw edema Treatment (mg/paw) (%) I-O 10 18 I-OE 10 19 I-OM 10 29 I-OA 1025 II-O 10 18 II-OW 10  0 II-OM 10 26 II-OA 10 25 II-OC 10  8 III-O 10 0 III-OM 10 11 III-OA 10 15 [6]-dehydrogingedione  5 26 1. Inhibitoryactivity on the carrageenin-induced paw edema (%) was calculated asfollows: [(average degree of edema of mice in the control group) −(average degree of edema of mice in the test group)/(average degree ofedema of mice in the control group)] × 100% 2. Values are presented asmean ± S.E., n = 3-6.

What is claimed is:
 1. A method of preparing a product potent inanti-inflammation or in anti-platelet aggregation from rhizomes ofZingiber officinale comprising the following steps: a) preparing a crudeliquid from rhizomes of Zingiber officinale; b) introducing the crudeliquid to a reverse phase chromatography column, and eluting the columnwith water, a first eluent and a second eluent in sequence, said secondeluent having a polarity weaker than that of the first eluent butstronger than that of chloroform, so that a first eluate resulting fromelution of the first eluent and a second eluate resulting from elutionof the second eluent are obtained; c) removing the first eluent from thefirst eluate by evaporation, so that a first concentrated eluate isobtained and is able to be used as the potent product; and d) removingthe second eluent from the second eluate by evaporation, so that asecond concentrated eluate is obtained and is able to used as the potentproduct; wherein step a) comprises steps i) to iv), or comprises stepI), step I′), or step I″), wherein said steps i) to iv) are: i) sheddingfresh rhizomes of Zingiber officinale and filleting the resultingmixture to obtain a filtrate and a residue; ii) extracting the filtratewith a first organic solvent, recovering the resulting extractionsolution of the first organic solvent, and evaporating the first organicsolvent from the extraction solution to obtain a first concentratedextraction solution; iii) extracting the residue with a second organicsolvent, recovering the resulting extraction solution of the secondorganic solvent, and evaporating the second organic solvent from theextraction solution to obtain a second concentrated extraction solution;and iv) combining the first concentrated extraction solution and thesecond concentrated extraction solution to obtain the crude liquid; saidstep I) is: I) extracting powder of dried rhizomes of Zingiberofficinale with the second organic solvent, recovering the resultingextraction solution of the second organic solvent, and evaporating thesecond organic solvent from the extraction solution to obtain the crudeliquid; said step I′) is: I′) steam distilling powder of dried rhizomesof Zingiber officinale, and concentrating the resulting distillate byevaporation to obtain the crude liquid; and said step I″) is: I″)extracting powder of dried rhizomes of Zingiber officinale withsupercritical CO₂, recovering the resulting extraction solution of thesupercritical CO₂, and evaporating CO₂ from the extraction solution toobtain the crude liquid.
 2. The method according to claim 1, wherein theproduct potent in anti-inflammation or in anti-platelet aggregationcomprises 0-10 mg 6-shogaol per gram of the product, 1-150 mg 6-gingerolper gram of the product, and 0-40 mg 6-dehydrogingerdione per gram ofthe product.
 3. The method according to claim 1, wherein said firsteluent is methanol, and said second eluent is acetone.
 4. The methodaccording to claim 3, wherein step a) comprises steps i) to iv).
 5. Themethod according to claim 4, wherein said first organic solvent is ethylether.
 6. The method according to claim 4, wherein said second organicsolvent is acetone, methanol, ethanol or a combination of them.
 7. Themethod according to claim 6, wherein said second organic solvent isacetone.
 8. The method according to claim 3, wherein step a) comprisesstep I).
 9. The method according to claim 8, wherein said second organicsolvent is acetone, methanol, ethanol or a combination of them.
 10. Themethod according to claim 9, wherein said second organic solvent isacetone.
 11. The method according to claim 3, wherein step a) comprisesstep I′).
 12. The method according to claim 3, wherein step a) comprisesstep I″).
 13. The method according to claim 3, wherein said reversephase chromatography column is packed with a porous resin.
 14. Ananti-inflammation pharmaceutical composition comprising atherapeutically effective amount of a crude liquid prepared in step a)of the method according to claim 1, as an active ingredient, inadmixture with a pharmaceutically acceptable carrier or diluent for theactive ingredient.
 15. The pharmaceutical composition according to claim14, wherein step a) comprises steps i) to iv).
 16. The pharmaceuticalcomposition according to claim 15, wherein said first organic solvent isethyl ether.
 17. The pharmaceutical composition according to claim 16,wherein said second organic solvent is acetone, methanol, ethanol or acombination of them.
 18. The pharmaceutical composition according toclaim 17, wherein said second organic solvent is acetone.
 19. Thepharmaceutical composition according to claim 14, wherein step a)comprises step I).
 20. The pharmaceutical composition according to claim19, wherein said second organic solvent is acetone, methanol, ethanol ora combination of them.
 21. The pharmaceutical composition according toclaim 20, wherein said second organic solvent is acetone.
 22. Thepharmaceutical composition according to claim 14, wherein step a)comprises step I′).
 23. The pharmaceutical composition according toclaim 14, wherein step a) comprises step I″).
 24. A pharmaceuticalcomposition for the inhibition of aggregation of platelet, whichcomprises a therapeutically effective amount of the first concentratedeluate prepared in step c) of the method according to any one of claims1 to 13, as an active ingredient, in admixture with a pharmaceuticallyacceptable carrier or diluent for the active ingredient.
 25. Ananti-inflammation pharmaceutical composition comprising atherapeutically effective amount of the first concentrated eluateprepared in step c) of the method according to any one of claims 1 to13, as an active ingredient in admixture with a pharmaceuticallyacceptable carrier or diluent for the active ingredient.
 26. Ananti-inflammation pharmaceutical composition comprising atherapeutically effective amount of the second concentrated eluateprepared in step d) of the method according to any one of claims 1 to13, as an active ingredient, in admixture with a pharmaceuticallyacceptable carrier or diluent for the active ingredient.
 27. Apharmaceutical composition for the inhibition of aggregation ofplatelet, which comprises a therapeutically effective amount of saidcrude liquid prepared in step a) of the method according to claim 1, asan active ingredient, in admixture with a pharmaceutically acceptablecarrier or diluent for the active ingredient.
 28. The pharmaceuticalcomposition according to claim 27, wherein step a) comprises steps i) toiv).
 29. The pharmaceutical composition according to claim 28, whereinsaid first organic solvent is ethyl ether.
 30. The pharmaceuticalcomposition according to claim 28, wherein said second organic solventis acetone, methanol, ethanol or a combination of them.
 31. Thepharmaceutical composition according to claim 30, wherein said secondorganic solvent is acetone.
 32. The pharmaceutical composition accordingto claim 27, wherein step a) comprises step I).
 33. The pharmaceuticalcomposition according to claim 32, wherein said second organic solventis acetone, methanol, ethanol or a combination of them.
 34. Thepharmaceutical composition according to claim 33, wherein said secondorganic solvent is acetone.
 35. The pharmaceutical composition accordingto claim 27, wherein step a) comprises step I′).
 36. The pharmaceuticalcomposition according to claim 27, wherein step a) comprises step I″).37. A pharmaceutical composition for the inhibition of aggregation ofplatelet, which comprises a therapeutically effective amount of thesecond concentrated eluate prepared in step d) of the method accordingto any one of claims 1 to 13, as an active ingredient, in admixture witha pharmaceutically acceptable carrier or diluent for the activeingredient.